PDIA3 inhibits mitochondrial respiratory function in brain endothelial cells and C. elegans through STAT3 signaling and decreases survival after OGD

Background Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. Methods Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen–glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. Results We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen–glucose deprivation, while STAT3 inhibition reduced the protective effect. Conclusions We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation. Supplementary Information The online version contains supplementary material available at 10.1186/s12964-021-00794-z.

Within mitochondria, PDIA3 promotes the intrinsic apoptotic pathway and mitochondrial permeabilization [6,19,20]. Furthermore, PDIA3 can increase mitochondrial calcium uptake by transcriptionally upregulating the mitochondrial calcium uniporter [21]. Finally, PDIA3 is present at ER-mitochondrial contact sites, suggesting a possible role for non-transcriptional regulation between these organelles [22]. A general protein disulfide isomerase inhibitor can protect against reductions in ATP levels in an in vitro Huntington disease model [12], supporting the notion that PDIA3 might inhibit mitochondrial bioenergetic function.
A potential mechanism through which PDIA3 might modulate mitochondrial bioenergetics is through its interaction with STAT3. PDIA3 is a chaperone for STAT3 and can modulate STAT3 transcriptional activity by inhibiting or promoting phosphorylation of the Y705 residue [8,9,[23][24][25] and inhibiting STAT3 S727 phosphorylation [23,26]. Phosphorylation of only the S727 residue targets STAT3 for mitochondrial import [27], where it promotes bioenergetic function and reduces reactive oxygen species (ROS) [28,29]. In cardiomyocytes, STAT3 prevents mitochondrial permeabilization through inhibiting mitochondrial permeability transition pore opening and reduces toxic ROS levels during ischemia/reperfusion injury [30,31]. Phosphorylated S727 (pS727) STAT3 also preserves mitochondrial function during post-ischemic conditioning after injury [32]. We have shown that reduced mitochondrial pS727 STAT3 in endothelial cells is associated with ER stress-induced cell death [33], while increased mitochondrial pS727 STAT3 improves bioenergetic function and endothelial cell survival [34]. Brain endothelial cells have very high concentrations of mitochondria [35,36], which are important for maintaining the blood-brainbarrier [37,38]. Here, we investigated whether PDIA3 suppresses mitochondrial bioenergetic function through STAT3 by using CMECs, a well-differentiated human brain endothelial cell line. We used C. elegans to test the role of the homologue, PDI3, in an intact animal because the PDIA3 knockout in mice is embyronically lethal [9]. Additionally, we tested whether PDIA3 deletion would be protective against OGD injury in cultured CMECs.

CMECs and C. elegans
CMECs are an immortalized clone of well-differentiated microvascular endothelial cells originally obtained from brain tissue of a female epilepsy patient [39,40] and were expanded and maintained at 37 °C and 5% CO 2 as described [41]. We chose to use CMEC's as these cells are highly transfectable which allows for genetic manipulation and brain endothelial cells have robust expression of PDIA3 [42]. N2 and TP66 C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC). C. elegans were chosen as a model because a knockout is available (TP66) while PDIA3 KO in mice leads to a lethal phenotype [9]. TP66 worms have a pdi-3 gene deletion, originally identified by genetic screens of mutants generated by random UV/trimethylpsoralen and were backcrossed four times [10]. TP66 worms were found to be homozygous for the pdi-3 deletion with genotyping performed by PCR of isolated genomic DNA (primers: 5′-CGT GTC TTG AAA GTT GCT C-3′, 3′-CCC TCT AAC TTC GAA CAT TGG-5′). No difference was observed between the strains in the developmental time from L1 larvae to adulthood. Worms were grown on Nematode Growth Media agar plates with a surface coating of live OP50 E. coli feeder bacteria [43]. The worms were synchronized by bleaching followed by overnight suspension in M9 buffer to obtain L1 stage larva and experiments were performed at the L3/L4 stage.

Bioenergetic measurements
Oxygen consumption rate (OCR) assays were performed as described [44] with minor modifications. Briefly, we used an XF HS mini-Seahorse analyzer (Agilent) with Flux Packs (103,022-100, Agilent). For quantification of bioenergetic functions, basal respiration was calculated as baseline OCR minus OCR level after antimycin A, ATP-linked respiration as the level after baseline minus oligomycin, reserve (spare) capacity as the level after FCCP minus baseline, and maximal respiration as the OCR level reached after FCCP minus the antimycin A level. CMECs were plated onto collagen type 1 (3440-005-01, Cultrex, 150 µg/ml in H 2 O) at ~ 20,000 cells/well. L3/4 stage worms were fed heat-killed OP50 E. coli bacteria for 48 h to avoid background respiration from E. coli that may have stuck to C. elegans after washing. Bacteria were heat killed by two cycles of flash freezing in liquid nitrogen followed by heating to 70 °C for 2 h. Immediately prior to analysis, worms were washed 5× with 0.1 M NaCl to remove excess bacteria before counting and plating ~ 75 worms/well in M9 medium. For worms, 40 mM sodium azide was used instead of antimycin A and oligomycin was excluded because it cannot readily diffuse through the worm cuticle. C. elegans were counted by an investigator who was blinded to the genotype and treatment of the worms from images taken after the assays were performed.

RT-qPCR
RT-qPCR was performed using Taqman probes as described [46]. Analyses were performed using the delta-delta-Ct method with relative gene expression first normalized to GAPDH mRNA and then expressed relative to the experimental controls. Primers for human PDIA3 (Hs04194196_g1) and GAPDH (Hs02786624_ g1) or mouse PDIA3 (Mm00433130_m1) and GAPDH (Mm99999915_g1) were from ThermoFisher and designed to cross exon borders to specifically target cDNA.

OGD
OGD was performed as described [50]. CMECs were plated at 200,000 cells/well and maintained for 48 h before a 24 h incubation in glucose-free deoxygenated DMEM in a hypoxia chamber (5% CO 2 in 95% nitrogen). Afterwards, Hoechst was added to the medium and CMECs were imaged with a 20 × objective for quantification with ImageJ software, using a grid set to 100,000pixel size, with nine squares per image counted blindly. Live cells were characterized by large diffuse nuclei and were counted blindly. Lactate dehydrogenase release was used as an indicator of cell death and measured according to manufacturer's protocol (786-210, G-Biosciences).

Statistics
Sample sizes were based upon power analysis (G*power) and indicate independent experiments in the results. Statistical analyses used GraphPad Prizm with Student's t-test for two groups, one-way ANOVA for more than two groups, and a two-way ANOVA for multiple variables in more than two groups with normally (gaussian) distributed data.

PDIA3 inhibits mitochondrial bioenergetic function in CMECs
PDIA3 is known to regulate STAT3 signaling but its role in bioenergetic function is unknown. Genetic deletion of PDIA3 in CMECs with CRISPR-Cas9 was confirmed by qPCR (Fig. 1a,   . WT CMECs were transfected with non-targeting gRNA. c Genotype was further confirmed by PCR from genomic CMEC DNA surrounding the target cut site. d Respiratory rates, as measured by oxygen consumption rate (OCR) by Seahorse analysis, were increased in PDIA3-/-CMECs relative to WT cells (repeated measures ANOVA. n = 3 per condition). e Reserve (spare) capacity (Res. Cap.) and maximal respiratory capacity (Max. Resp.) were increased but not basal respiratory rate and ATP-linked respiration (n = 3 each, two-way ANOVA). f The PDIA3-/-phenotype of increased bioenergetic function was largely reversed by overexpressing WT human PDIA3 compared to GFP control transfection (g) capacity and an increased ability to respond to stress or higher energy demand [51]. The maximum respiratory capacity was also increased in PDIA3-/-relative to control-transfected WT cells (Fig. 1e, n = 3, p < 0.01), indicating these cells have increased maximum respiratory rates. As a confirmation, PDIA3 overexpressed in PDIA3-/-CMECs suppressed bioenergetic function (Fig. 1f, n = 3, p < 0.001), particularly maximal respiratory capacity (Fig. 1g, n = 3, p < 0.01) relative to GFP-transfected controls.

PDI-3 inhibits bioenergetic function in C. elegans
We determined whether PDIA3 inhibited mitochondrial function in an evolutionarily distant species and in an intact multicellular organism. The pdi-3 gene is the C. elegans worm ortholog of mammalian PDIA3 and has ~ 50% amino acid homology and conserved catalytic thioredoxin domains (Fig. 2a). TP66 worms have a pdi-3 deletion [10] as confirmed by PCR showing a shorter amplicon due to a lesion around exon 3 [10] (Fig. 2b). OCR was increased in TP66 relative to WT N2 strain worms (Fig. 2c), showing increased reserve capacity and maximal respiration (Fig. 2d, n = 6 independent plates, p < 0.05). Moreover, RNAi knockdown of pdi-3 in WT N2 worms increased OCR, particularly maximal respiration, relative to control-transfected N2 worms (Fig. 2e).

PDIA3 reduces mitochondrial activity
MitoTracker Red CMXRos dye accumulation depends on mitochondrial membrane potential [52]. PDIA3-/-CMECs treated with this dye had greater red fluorescence than WT CMECs (Fig. 3a, n = 6, p < 0.001) when normalized to nuclear Hoechst stain to account for variability in cell numbers [33]. In contrast, the signal from LysoTracker Red dye, which fluoresces when localized to acidic organelles such as lysosomes and autophagosomes, was not different between genotypes (Fig. 3b, n = 6), suggesting that PDIA3-/-CMECs did not have alterations in lysosomal or autophagosomal mass or mitophagy [53]. PDIA3-/-cells had increased MitoSox Red fluorescence, indicating increased mitochondrial superoxide production (Fig. 3c, n = 4, p < 0.01), likely due to the increased mitochondrial electron transport chain activity as indicated by the by the increased OCRs.

PDIA3-/-CMECs have normal mitochondrial mass
To exclude the possibility that bioenergetic increases were caused by changes in mitochondrial mass, protein from purified mitochondria were quantified using capillary western analysis. Pyruvate dehydrogenase subunit A1 (PDHA1) is essential for metabolizing the end product of glycolysis into the mitochondrial citric acid cycle and altered PDH activity affects OCR [54]. PDHA1 expression was not different between PDIA3-/-and WT CMECs (Fig. 3d, e). Cytosolic GAPDH was not detected, confirming the purity of the mitochondrial fractions (Fig. 3f, g). PDIA3 was present in WT mitochondrial Fig. 3 Mitochondrial membrane potential and ROS, but not mitochondrial mass, is increased in CMECs. a MitoTracker Red CMXRos dye is a potentiometric dye that accumulates in active mitochondria. Fluorometry showed that MitoTracker Red CMXRos intensity was greater in PDIA3-/-CMECs relative to WT (~ 20%, p < 0.001, n = 4, Student's t-test). Values were normalized to nuclear Hoechst dye to account for potential variations in cell number and expressed as a fold of WT. b The fluorescence of LysoTracker Red, a dye that accumulates in lysosomes, was not different between genotypes. c Superoxide was measured using MitoSox Red. PDIA3-/-CMECs had higher levels of superoxide (n = 4, p < 0.01) relative to WT CMECs. d Synthetic bands derived from peak chemiluminescent intensity of e capillary western blotting of proteins from isolated mitochondria show that the amount of pyruvate dehydrogenase (PDHA1) was not different between PDIA3-/-and WT CMECs. The chemiluminescence peak amount is indicated by the number 55 K indicating 55,000. f, g GAPDH, a cytoplasmic protein, was largely absent, indicating the purity of the mitochondrial sample. h, i PDIA3 was present in mitochondrial fractions of WT, but not PDIA3-/-, CMECs. j, k Regular western blots of whole cell lysates show that the PDIA3-/-and WT CMECs have similar amounts of the mitochondrial protein PDHA1, indicating a similar mitochondrial mass. The cytoplasmic household protein α-tubulin was used as loading control extracts, but not those from PDIA3-/-CMECs (Fig. 3h, i), confirming PDIA3 is present in the mitochondria. Finally, PDHA1 was equally abundant in whole cell lysates of both genotypes, further suggesting that mitochondrial mass is not altered by PDIA3 knockout (Fig. 3j, k). Moreover, the mitochondrial tubular networks of MitoTracker Green-treated cells appeared similar in PDIA3-/-and WT CMECs, and their estimated mitochondrial surface area (Fig. 4b) and volume visualized by by confocal microscopy were not different (Fig. 4c).

PDIA3 promotes cell growth
Mitochondrial function is often linked to proliferation and survival [58], which we controlled for as a potential confounding factor in our analyses. PDIA3-/-CMECs grew more slowly between 48 and 72 h after plating (Additional file 1: Fig. S1A, B, p < 0.05, n = 3). Release of lactate dehydrogenase, a measure for cell death, was not different between genotypes between 48 and 72 h (Additional file 1: Fig. S1C). Thus, PDIA3 might promote proliferation, but not cell death, under normal conditions. The late differences in proliferation between PDIA3-/and WT CMECs should not confound the OCR measurements made 24 h after plating or compared within the same genotype. Moreover, the OCR data were normalized to protein content.

Discussion
Our most important and novel findings are that PDIA3 inhibits mitochondrial respiratory function, S727 STAT3 phosphorylation and cell survival under OGD conditions. The novel role of PDIA3 in downregulating mitochondrial bioenergetics is consistent with proteomics data indicating its potential involvement in modulating aerobic capacity in skeletal muscle [26]. PDIA3 had been shown to inhibit STAT3 S727 phosphorylation and our genetic and pharmacological results demonstrate that this downregulates mitochondrial respiratory function and cell survival under OGD conditions [23]. STAT3 regulation of mitochondrial respiration is dependent upon S727 phosphorylation and subsequent interaction with retinoid-interferon mortality-19 (GRIM19), which mediates mitochondrial import and integration with complex I of the electron transport chain [27,55]. Thus, loss of PDIA3 and consequently increased pS727 may lead to increased availability of STAT3 for mitochondrial import.
Although PDIA3 binds to STAT3 by direct protein-protein interaction [23] and modulates its signaling [61,62], the mechanism by which it suppresses pS727 is unclear, as PDIA3 is not a phosphatase. PDIA3 is known to chaperone proteins [7] and it might act as a chaperone for serine phosphatases that dephosphorylate S727 STAT3 and bring them in close proximity to STAT3 S727. PDIA3-STAT3 association typically occurs in either the cytosol [63] or the nucleus [8]. However, like others [20], we found PDIA3 within mitochondria as well, suggesting that PDIA3 could promote dephosphorylation of pS727 within mitochondria. We have shown that STAT3 S727 is dephosphorylated in isolated mitochondria, indicating the presence of mitochondrial-resident serine phosphatases that can perform this function [34]. It remains to be determined whether PDIA3 exerts its observed influence on mitochondrial respiration mostly from its binding to complex I on the inner membrane of the organelle or indirectly from elsewhere. PDIA3 might also contribute to cell survival and death through other pathways that affect mitochondria. Depletion of the related PDIA1 in endothelial cells causes increased mitochondrial fission and ROS production, Fig. 5 PDIA3 inhibits mitochondrial function by suppressing pS727 STAT3. a STAT3 S727 phosphorylation was increased in PDIA3-/-CMECs, as confirmed by densitometry (b), * p < 0.05, Student's t-test). Phosphorylation of STAT3 Y705 was not obviously different in PDIA3-/-CMECs, considering the total amounts of STAT3 and the β-actin loading control. We previously found that FAK drives mitochondrial bioenergetics via STAT3 S727 phosphorylation [34] but pFAK was not different in PDIA3-/-(KO) CMECs, as confirmed by densitometry (c). d Overexpression of WT human PDIA3 in PDIA3-/-CMECs reduced pSTAT3 S727, as seen by western blotting and densitometry (e). f) The inhibitory effect of PDIA3 on STAT3 S727 phosphorylation was confirmed by siRNA mediated knockdown of PDIA3 (siPDIA3) in WT CMECs relative to a non-targeting control siRNA (siCtrl). Knockdown increased STAT3 S727 phosphorylation (g). h Finally, we tested whether increased STAT3 S727 phosphorylation was necessary for PDIA3-/-increased respiratory function. Inhibition of STAT3 with stattic in PDIA3-/-CMECs, reduced respiratory function compared within 1 h, as measured by Seahorse assay (n = 6, p < 0.01, one-way ANOVA). i Quantification showed decreased basal and ATP-linked respiration (n = 6, p < 0.01, two-way ANOVA), which indicates PDIA3 suppresses mitochondrial function through inhibition of STAT3 leading to cellular senescence [64], although bioenergetic function was not tested. Mitochondrial fragmentation and increases in ROS have been observed in hyperglycemic conditions [65]. A potential role of PDIA3 in decreasing mitophagy is suggested by its role in autophagy in pancreatic beta cells leading to apoptosis [66]. Here, PDIA3 deletion did not affect mitochondrial mass or morphology, but, like depletion of PDIA1, it increased ROS production. Thus, increased bioenergetics upon PDIA3 deletion in CMECs and C. elegans is likely the result of increases in the activity of key electron transport chain complexes. Vitamin D restores autolysosomal function in gut epithelial cells through activation of PDIA3 [24]. Although changes in mitophagy and mitochondrial turnover could result in altered bioenergetic capacity, we did not detect differences in lysosomal content between WT and PDIA3-/-CMECs. On the other hand, PDIA1, like PDIA3, contains the prototypical -CGHC-thioredoxin amino acid sequence at its amino and carboxyl termini, which can reduce ROS accumulation. Loss of either one may reduce ROS scavenging capacity within a cell. In our study, PDIA3-/-CMECs showed an increased mitochondrial membrane potential, as well as greater ROS production. This is consistent with a role for PDIA3 as an inhibitor of bioenergetic capacity.
Our study also suggests that PDIA3 inhibition of mitochondrial function is not restricted to mammalian cells. In C. elegans, a homolog of mammalian STAT proteins STA-1 has only 30% homology to STAT3 and no data exists on whether PDI-3 interacts with STA-1 [67]. The shortened (dumpy) morphological phenotype of pdi-3 deleted worms [10] is likely caused by decreased collagen crosslinking and extracellular matrix stabilization due to the lack of PDI-3 transglutaminase activity, as well as the loss of chaperone function in the ER. However, the dumpy phenotype may be enhanced by the increased respiration which produces increased amounts of ROS that are known to modulate cuticle remodeling during molting [68,69]. PDIA3 may also play a role in suppressing muscle cell functions in which PDIA3 has a potential involvement in aerobic capacity [26]. PDIA3 expression is increased in the gastrocnemius muscle of 30 month old relative to younger 7 month old rats [70] and in chronological muscle ageing in post-menopausal women [71], potentially explaining in part why aged skeletal muscle has reduced oxidative capacity relative to young muscle [72].
Our data also suggest that PDIA3 plays a detrimental role under pathological conditions such as OGD, which mimics key aspects of ischemia. Importantly, both OGD and ischemia are characterized by mitochondrial bioenergetic failure [33]. Long-term bioenergetic dysfunction leads to apoptosis [73]. PDIA3 protein expression is upregulated after OGD in SHSY-5Y neuronal cells [74], but was not altered in the CMECs. Mitochondria play a key role in apoptosis [73], releasing cytochrome C through the mitochondrial permeability transition pore, which STAT3 can suppress [75]. PDIA3 is known to stabilize expression of mitochondrial calpains and cleavage of apoptosis inducing factor, thereby contributing to increased apoptosis [6], so abrogation of PDIA3 functions could result in decreases in pro-apoptotic pathway activation. In addition to this, PDIA3 interacts directly with STAT3 to suppress pY705 [23]. It is likely that during this interaction, pS727 is inhibited [76]. By reducing the amount of pSTAT3 Y705 in the nucleus, PDIA3 may increase the available pool of cytoplasmic STAT3 capable of being phosphorylated on S727 and, after translocation to the mitochondria, promoting mitochondrial function and cell survival. Alternatively, reduced PDIA3 can function as a ROS scavenger [77], and increased ROS promotes STAT3 phosphorylation on S727 and mitochondrial recruitment [78]. Thus, the ROS increases observed in PDIA3-/-cells could be the mechanistic link between PDIA3 and pSTAT3 In endothelial cells, PDIA3 inhibits phosphorylation of the S727 residue of STAT3, which promotes mitochondrial bioenergetics. PDIA3 gene knockout with CRISPR or knockdown with siRNA increases STAT3 activation, mitochondrial bioenergetics, cellular function and cell survival under OGD conditions. In C. elegans, the PDI-3 homolog plays a similar role in mitochondrial bioenergetics as shown in the TP66 mutant strain S727. Following OGD, we found that pY705 was completely abolished in both WT and PDIA3-/-CMECs, suggesting that Y705 is not important for cell survival in this model. Pharmacological inhibition of STAT3 with stattic resulted in a loss of PDIA3-/-protective effects against OGD. Other studies found that inhibition of STAT3 can be protective against hypoxic injury by blocking translocation to the nucleus [79]. STAT3 has also been found in the ER, where it has a role in regulating calcium homeostasis as key to its anti-apoptotic activities [76]. We observed increased mitochondrial superoxide in PDIA3-/-cells and this may lead to decreased rates of cell growth (Additional file 1: Fig. S1) through downregulation of cyclins involved in cell cycle checkpoints. P21 and P53 are upregulated in response to ROS production, delaying progression from G1 to S phases of the cell cycle [80]. At the same time, it is possible that loss of PDIA3 leads to decreased cell death through increased STAT3 S727 phosphorylation and reduced calcium overload [76]. Further, reduced FAK-S727 STAT3 activation following ER stress in mouse bEnd5 endothelial cells leads to decreased cell survival [33], suggesting an important role for this pathway in preventing apoptosis under injurious conditions. Here, both FAK pY397 and STAT3 pS727 were reduced following OGD, suggesting that partial preservation of this signaling pathway protects these cells. However, the mechanism for how PDIA3-/-increases FAK pY397 under OGD, but not under normoxic conditions requires further investigation.

Conclusions
In conclusion, our study defines a novel role for PDIA3 in inhibiting mitochondrial bioenergetics (Fig. 7), whereby decreases in PDIA3 increases respiratory function and reduces cell loss during OGD. This presents an important step forward in understanding PDIA3 functions related to STAT3 signaling and new avenues for therapeutic intervention for diseases such as stroke.